Recombinant human alpha-1-antitrypsin for the treatment of inflammatory disorders

ABSTRACT

In one aspect, the disclosure relates to compositions comprising alpha-1-antitrypsin (AAT) and the production thereof. In some embodiments, the AAT is recombinantly produced. The disclosure also relates to methods of administering compositions comprising alpha-1-antitrypsin (AAT).

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional application 61/577,289, filed Dec. 19, 2011, the entirecontents of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the treatment of inflammatoryconditions including asthma, emphysema, chronic obstructive pulmonarydisease and chronic granulomatous lung disease i.e., sarcoid. Inparticular, the invention relates to treatment of these conditions usingrecombinant human alpha-1-antitrypsin.

BACKGROUND OF THE INVENTION

Recombinant proteins provide effective therapies for manylife-threatening diseases. The use of high expression level systems suchas bacterial, yeast and insect cells for production of therapeuticprotein is limited to small proteins without extensivepost-translational modifications. Mammalian cell systems, whileproducing many of the needed post-translational modifications, are moreexpensive due to the complex, and, therefore, sophisticated culturesystems that are required. Moreover, in these sophisticated cell culturemethods reduced protein expression levels are often seen. Some of thelimitations of mammalian cell culture systems have been overcome withthe expression of recombinant proteins in transgenic mammals or avians.Proteins have been produced in mammary glands of various transgenicanimals with expression levels suitable for cost effective production atthe scale of hundreds of kilograms of protein per year.

SUMMARY OF THE INVENTION

In one aspect the disclosure provides recombinant humanalpha-1-antrypsin (AAT). In one aspect, recombinantly producedrecombinant human alpha-1-antrypsin (AAT) is administered to a patientin need of AAT.

Unexpectedly, it was found that the administration of recombinant humanalpha-1 antitrypsin (AAT) provides higher efficacy in the lung than acorresponding dosage of plasma derived AAT. Without being bound by anyspecific theory, it is believed that the glycosylation profile ofrecombinant AAT produced in the milk of transgenic goats provides anincreased localization of the protein in the lung compared to that ofplasma derived AAT.

In one aspect, the disclosure provides a composition comprisingalpha-1-antitrypsin (AAT), wherein the AAT is recombinantly produced. Insome embodiments, the AAT is produced in mammary epithelial cells of anon-human mammal. In some embodiments, the AAT is produced in atransgenic non-human mammal. In some embodiments, the non-human mammalis a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat,mouse or llama. In some embodiments, the non-human mammal is a goat. Insome embodiments, the recombinantly produced AAT has enhanceddeoxyhexose glycosylation compared to plasma-derived AAT. In someembodiments, the recombinantly produced AAT has been modified toincrease the sialylation on the AAT-glyco-motifs.

In one aspect, the disclosure provides a composition comprising AATwherein the AAT has a high level of deoxyhexose glycosylation. In oneaspect, the disclosure provides a composition comprising AAT wherein theAAT has a high level of sialylation on the AAT-glyco-motifs. In oneaspect, the disclosure provides a composition comprising AAT wherein theAAT has a high level of deoxyhexose glycosylation and a high level ofsialylation on the AAT-glyco-motifs.

In some embodiments of any of the compositions of AAT described herein,the composition further comprises milk. In some embodiments of any ofthe compositions of AAT described herein, the composition furthercomprises a pharmaceutically acceptable carrier.

In one aspect, the disclosure provides mammary gland epithelial cellsthat produce the AAT of the compositions of any of the compositionsdescribed herein. In one aspect, the disclosure provides a transgenicnon-human mammal comprising the mammary gland epithelial cells disclosedherein.

In one aspect, the disclosure provides methods of administering the AATcompositions disclosed herein to a subject in need thereof. In someembodiments, the subject has alpha-1-antitrypsin deficiency. In someembodiments, the subject has an inflammatory disorder. In someembodiments, the inflammatory disorder is emphysema. In someembodiments, the composition is administered at a dose of from 30 mg/kgto about 60 mg/kg AAT. In some embodiments, the composition isadministered intravenously. In some embodiments the composition isadministered by inhalation.

In one aspect, the disclosure provides a method of reducing elastaseactivity in the lung, the method comprising administering the AATcompositions disclosed herein to a subject in an amount sufficient toreduce elastase activity in the lung.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are exemplary and not required for enablement of theinvention.

FIG. 1 shows Coomassie staining of rat broncholalveolar lavage (BAL)samples (FIG. 1A) and quantification of the staining of AAT harvestedfrom BAL (FIGS. 1B and 1C). The samples are normalized based on albuminharvested from BAL.

FIG. 2 shows the level of GRO/CINC-1 ELISA (Growth-regulated geneproduct/cytokine-induced neutrophil chemoattractant) which is correlatedwith IL-8 and a model of inflammation in broncholalveolar lavage samplesof rats treated with AAT.

FIG. 3 shows the elastase reactivity of control AAT (lanes 2-5) andbroncholalveolar lavage harvested AAT (lanes 7-10). Binding to elastaseis indicated by an increase in molecular weight of the AAT.

FIGS. 4 A and B shows the amount as assayed by SDS page of AAT harvestedfrom the broncholalveolar lavage of rats which were administered a doseof 30 mg/kg of AAT.

FIG. 5 shows the amount as assayed by DOT blot analysis of AAT harvestedfrom the broncholalveolar lavage of rats which were administered a doseof 30 mg/kg of AAT.

FIG. 6 shows the pharmacokinetics of AAT in rats exposed to 30 mg//kg ofAAT.

FIG. 7 shows the relative level of AAT in blood vs. lung in rats thatwere administered 3 mg/kg AAT or 30 mg/kg AAT.

FIG. 8 shows the glycosylation pattern of recombinantly produced AAT.

FIG. 9 shows the glycosylation pattern of plasma-derived AAT.

FIGS. 10 A and B shows the amount as assayed by SDS page of AATharvested from the broncholalveolar lavage of rats which wereadministered a dose of 3 mg/kg of AAT

FIG. 11 shows the amount as assayed by DOT blot analysis of AATharvested from the broncholalveolar lavage of rats which wereadministered a dose of 3 mg/kg of AAT.

FIG. 12 shows the level of GRO/CINC-1 ELISA (Growth-regulated geneproduct/cytokine-induced neutrophil chemoattractant) which is correlatedwith IL-8 and a model of inflammation in broncholalveolar lavage samplesof rats treated with AAT.

FIG. 13 shows the analysis of BAL from 30 mg/kg exposed rats.

FIG. 14 shows the analysis of BAL from 3 mg/kg exposed rats.

FIG. 15 shows the pharmacokinetics of 30 mg//kg rat lung study.

FIG. 16 shows the pharmacokinetics of 3 mg//kg rat lung study.

FIG. 17 shows anti-elastase activity. times

FIG. 18 shows the results and design of an experiment according to themethods provided herein.

DETAILED DESCRIPTION

In one aspect the disclosure provides compositions comprisingrecombinantly produced Alpha-1-antitrypsin (AAT) and methods ofadministering recombinantly produced AAT to a subject in need thereof.

Alpha-1-antitrypsin is a glycoprotein with a molecular weight of 53,000,as determined by sedimentation equilibrium centrifugation. Theglycoprotein consists of a single polypeptide chain to which severaloligosaccharide units (glyco-motifs) are covalently bonded. Human alpha1-proteinase inhibitor has a role in controlling tissue destruction byendogenous serine proteinases. AAT is a suicide inhibitor that works byforming a stable tetrahedral intermediate with an enzyme, predominantlyelastase, after binding. Completion of the cleavage reaction isdependent on hydrolysis of both the C-terminal peptide (leaving group)and the active site serine. In most cases, the first hydrolysis takesplace and the enzyme is translocated across the beta sheet and“smashed”, disrupting the active site and rendering the enzyme inactiveand unable to complete the second hydrolysis, which leaves the enzymetethered to the AAT. If the second hydrolysis does occur, the AAT isreleased from the enzyme, minus its 36 amino acid peptide.Alpha-1-proteinase inhibitor inhibits human pancreatic and leukocyteelastases. See e.g., Pannell et al., Biochemistry 13, 5339 (1974);Johnson et al., Biochem Biophys Res Comm, 72 33 (1976); Del Mar et al.,Biochem Biophys Res Commun, 88, 346 (1979); and Heimburger et al., Proc.Int. Res. Conf. Proteinase Inhibitors 1^(st), 1-21 (1970).

A genetic deficiency of alpha-1-proteinase inhibitor, which accounts for90% of the trypsin inhibitory capacity in blood plasma, has been shownto be associated with the premature development of pulmonary emphysema.The degradation of elastin associated with emphysema probably resultsfrom a local imbalance of elastolytic enzymes and the naturallyoccurring tissue and plasma proteinase inhibitors. Currently, subjectsdeficient in AAT are treated with therapeutic concentrates ofalpha-1-antitrypsin prepared from the blood plasma of blood donors(plasma-derived AAT).

In one aspect, the disclosure provides methods of administering acomposition comprising recombinantly produced AAT to a subject in needthereof. In one aspect, the disclosure provides methods of reducingelastase activity in the lung, the method comprising administering acomposition comprising recombinantly produced AAT to a subject in anamount sufficient to reduce elastase activity in the lung.

Unexpectedly, it was found herein that recombinantly produced AAT(rhAAT) e.g., AAT produced in transgenic animals, upon administration issequestered in the lung at higher levels than a corresponding dose ofplasma derived AAT. As shown herein, rats were dosed with plasma-derivedAAT, rhAAT or sialylated rhAAT, and in these rats rhAAT and sialylatedrhAAT were found in the bronchial alveolar lavage (BAL) fluid at greaterlevels than plasma derived AAT. This sequestration into the lung waseven more surprising because of the lower concentrations of rhAAT andsialylated rhAAT in the blood. For instance, as shown herein, two hoursafter administration, recombinantly produced AAT is present in BAL at aconcentration approximately three times higher than the concentration ofplasma-derived AAT. This is even more remarkable if taken into accountthat the concentration of recombinantly produced AAT in the blood atthat same time is about six times lower than concentration ofplasma-derived AAT. The concentration of recombinantly produced AAT inthe blood is lower likely due to the higher clearance rate in the bloodof recombinantly produced AAT compared to plasma-derived AAT. The effectof sequestration in the lung is even more pronounced when sialylatedrecombinant AAT is compared to plasma-derived AAT. Sialylated AAT has alower clearance rate than unsialylated AAT and can therefore maintain ahigher level of recombinant AAT in the system.

It is also shown herein that the recombinant AAT harvested from BAL canbind elastase and it thus remains effective in the treatment of lungdisease. Furthermore, the recombinant AAT sequestered into the lung doesnot cause any more inflammation than found in a control experiment.Recombinantly produced AAT therefore has unexpected properties that makeit well suited for the treatment of lung disorders and/or inflammatorydisorders.

It should be appreciated that the AAT to be administered to a subjectshould generally be species-appropriate. In other words, if AAT is to beadministered to a human, the AAT will likely be human AAT. However, AATfrom other species may be administered (e.g., pig AAT administered to ahuman) as long as the AAT from a different species can still fulfill itsbiological role (e.g., bind human elastase) and does not cause aninappropriate immune response.

In one aspect, the disclosure provides compositions of recombinantlyproduced AAT, wherein the recombinantly produced AAT has enhanceddeoxyhexose glycosylation compared to plasma-derived AAT. In one aspect,the disclosure provides compositions of recombinantly produced AAT,wherein the recombinantly produced AAT has been modified to increase thesialylation on the AAT-glyco-motifs.

Recombinantly produced AAT has the same amino acid sequence asplasma-derived AAT. However, recombinantly produced AAT has aglycosylation pattern that is different from (human) plasma-derived AAT,as shown in the experimental section. In some embodiments, therecombinant AAT is produced in non-human mammary epithelial cells. Therecombinant AAT produced in non-human mammary epithelial cells has aglycosylation pattern that is determined inter alia by the prevalenceand interaction of glycosylation enzymes present in these mammaryepithelial cells.

While not being limited to a specific mechanism, it is assumed thatrecombinantly produced AAT is sequestered in the lung because it has ahigher affinity than plasma-derived AAT for glyco-receptors present inthe lung (receptors that bind the AAT glycoprotein and/or theglyco-motifs of the AAT glyco-protein). Again, while not being limitedto a specific mechanism the small amount of exposed N-acetylglucosaminepresent on recombinant AAT, which can bind the mannose receptor presentin the lung, may be responsible for the accumulation of recombinant AATin the lung. Alternatively, or in addition, deoxyhexose, which ispresent in larger amounts in the glyco motifs of recombinantly producedAAT than in plasma-derived AAT, may be responsible for the sequesteringin the lung.

In one aspect the disclosure provides a composition comprising AATwherein the AAT has a high level of deoxyhexose glycosylation. In oneaspect the disclosure provides a composition comprising AAT with a highlevel of sialylation on the AAT-glyco-motifs. In one aspect thedisclosure provides a composition comprising AAT wherein the AAT has ahigh level of deoxyhexose glycosylation and a high level of sialylationon the AAT-glyco-motifs.

It should further be appreciated that AAT that has a glycosylationpattern that is the same as the glycosylation pattern of recombinantlyproduced AAT can also be used in the methods described herein. Thus, insome embodiments the disclosure provides compositions and methods forthe administration of AAT that is not recombinantly produced, but thathas the same glycosylation pattern as recombinantly produced AAT. Thus,in some embodiments, the disclosure provides compositions and methods ofadministration of AAT comprising exposed N-acetylglucosamine. In someembodiments, the disclosure provides compositions and methods ofadministration of AAT comprising a high level of deoxyhexoseglycosylation. In some embodiments, a high level of deoxyhexoseglycosylation as used herein refers to a level of deoxyhexoseglycosylation that is 1.1 times or more, 1.2 times or more, 1.3 times ormore, 1.5 times or more, 2 times or more, 5 times or more, 10 times ormore, 50 times or more, or 100 times or more than the level ofdeoxyhexose glycosylation found in plasma-derived AAT. In someembodiments, a high level of deoxyhexose glycosylation as used hereinrefers to a population of AAT wherein at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% up to 100% of the glyco-motifsinclude a deoxyhexose moiety. In some embodiments, the disclosureprovides compositions and methods of administration of AAT comprising ahigh level of sialylation on the ATT glyco-motifs. In some embodiments,a high level of sialylation on the ATT glyco-motifs as used hereinrefers to a population of AAT wherein at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% up to 100% of the glyco-motifs ina population of AAT are sialylated.

Methods of modifying the glycosylation motif of a glycoprotein such asAAT are known in the art. For instance, a plasma-derived or E.coli-produced AAT can be subjected to enzymatic treatment with one ormore glycosylation enzymes to increase the amount of N-acetylglucosamineand/or deoxyhexose. For instance, treatment of AAT with neuraminidasefollowed by beta galactosidase may increase the amount of exposedN-acetylglucosamine.

Non-Human Mammary Gland Epithelial Cells for the Production of AAT

In one aspect, the disclosure provides mammary gland epithelial cellsthat produce AAT. In one aspect, the disclosure provides a transgenicnon-human mammal that produces AAT. In one aspect, the disclosurerelates to mammalian mammary epithelial cells that produce AAT. Methodsare provided herein for producing glycosylated AAT in mammalian mammaryepithelial cells. This can be accomplished in cell culture by culturingmammary epithelial cell (in vitro or ex vivo). This can also beaccomplished in a transgenic animal (in vivo).

In some embodiments, the mammalian mammary gland epithelial cells are ina transgenic animal. In some embodiments, the mammalian mammary glandepithelial cells have been engineered to express AAT in the milk of atransgenic animal, such as a mouse or goat. To accomplish this, theexpression of the gene(s) encoding the recombinant protein can be, forexample, under the control of the goat β-casein regulatory elements.Expression of recombinant proteins in both mice and goat milk has beenestablished previously (see, e.g., US Patent ApplicationUS-2008-0118501-A1). In some embodiments, the expression is optimizedfor individual mammary duct epithelial cells that produce milk proteins.

Transgenic animals capable of producing recombinant AAT can be generatedaccording to methods known in the art (see, e.g., U.S. Pat. No.5,945,577 and US Patent Application US-2008-0118501-A1) such methods areincorporated herein. Animals suitable for transgenic expression,include, but are not limited to goat, sheep, bison, camel, cow, pig,rabbit, buffalo, horse, rat, mouse or llama. Suitable animals alsoinclude bovine, caprine, ovine and porcine, which relate to variousspecies of cows, goats, sheep and pigs (or swine), respectively.Suitable animals also include ungulates. As used herein, “ungulate” isof or relating to a hoofed typically herbivorous quadruped mammal,including, without limitation, sheep, swine, goats, cattle and horses.Suitable animals also include dairy animals, such as goats and cattle,or mice. In some embodiments, the animal suitable for transgenicexpression is a goat.

In one embodiment, transgenic animals are generated by generation ofprimary cells comprising a construct of interest followed by nucleartransfer of primary cell nuclei into enucleated oocytes. Primary cellscomprising a construct of interest are produced by injecting ortransfecting primary cells with a single construct comprising the codingsequence of a protein of interest, e.g., AAT. These cells are thenexpanded and characterized to assess transgene copy number, transgenestructural integrity and chromosomal integration site. Cells withdesired transgene copy number, transgene structural integrity andchromosomal integration sites are then used for nuclear transfer toproduce transgenic animals. As used herein, “nuclear transfer” refers toa method of cloning wherein the nucleus from a donor cell istransplanted into an enucleated oocyte.

Coding sequences for AAT to be expressed in mammalian mammary epithelialcells can be obtained by screening libraries of genomic material orreverse-translated messenger RNA derived from the animal of choice (suchas humans, cattle or mice), from sequence databases such as NCBI,Genbank, or by obtaining the sequences by using methods known in theart, e.g. peptide mapping. The sequences can be cloned into anappropriate plasmid vector and amplified in a suitable host organism,like E. coli. As used herein, a “vector” may be any of a number ofnucleic acids into which a desired sequence may be inserted byrestriction and ligation for transport between different geneticenvironments or for expression in a host cell. Vectors are typicallycomposed of DNA although RNA vectors are also available. Vectorsinclude, but are not limited to, plasmids and phagemids. A cloningvector is one which is able to replicate in a host cell, and which isfurther characterized by one or more endonuclease restriction sites atwhich the vector may be cut in a determinable fashion and into which adesired DNA sequence may be ligated such that the new recombinant vectorretains its ability to replicate in the host cell. An expression vectoris one into which a desired DNA sequence may be inserted by restrictionand ligation such that it is operably joined to regulatory sequences andmay be expressed as an RNA transcript. Vectors may further contain oneor more marker sequences suitable for use in the identification of cellswhich have or have not been transformed or transfected with the vector.Markers include, for example, genes encoding proteins which increase ordecrease either resistance or sensitivity to antibiotics or othercompounds, genes which encode enzymes whose activities are detectable bystandard assays known in the art (e.g., β-galactosidase or alkalinephosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques. Afteramplification of the vector, the DNA construct can be excised, purifiedfrom the remains of the vector and introduced into expression vectorsthat can be used to produce transgenic animals. The transgenic animalswill have the desired transgenic protein integrated into their genome.

A DNA sequence which is suitable for directing production to the milk oftransgenic animals can carry a 5′-promoter region derived from anaturally-derived milk protein. This promoter is consequently under thecontrol of hormonal and tissue-specific factors and is most active inlactating mammary tissue. In some embodiments the promoter used is amilk-specific promoter. As used herein, a “milk-specific promoter” is apromoter that naturally directs expression of a gene in a cell thatsecretes a protein into milk (e.g., a mammary epithelial cell) andincludes, for example, the casein promoters, e.g., α-casein promoter(e.g., alpha S-1 casein promoter and alpha S2-casein promoter), β-caseinpromoter (e.g., the goat beta casein gene promoter (DiTullio,BIOTECHNOLOGY 10:74-77, 1992), γ-casein promoter, κ-casein promoter,whey acidic protein (WAP) promoter (Gorton et al., BIOTECHNOLOGY 5:1183-1187, 1987), β-lactoglobulin promoter (Clark et al., BIOTECHNOLOGY7: 487-492, 1989) and α-lactalbumin promoter (Soulier et al., FEBSLETTS. 297:13, 1992). Also included in this definition are promotersthat are specifically activated in mammary tissue, such as, for example,the long terminal repeat (LTR) promoter of the mouse mammary tumor virus(MMTV). In some embodiments the promoter is a caprine beta caseinpromoter.

The promoter can be operably linked to a DNA sequence directing theproduction of a protein leader sequence which directs the secretion ofthe transgenic protein across the mammary epithelium into the milk. Asused herein, a coding sequence and regulatory sequences (e.g., apromoter) are said to be “operably joined” or “operably linked” whenthey are linked in such a way as to place the expression ortranscription of the coding sequence under the influence or control ofthe regulatory sequences. As used herein, a “leader sequence” or “signalsequence” is a nucleic acid sequence that encodes a protein secretorysignal, and, when operably linked to a downstream nucleic acid moleculeencoding a transgenic protein, directs secretion. The leader sequencemay be the native human leader sequence, an artificially-derived leader,or may be obtained from the same gene as the promoter used to directtranscription of the transgene coding sequence, or from another proteinthat is normally secreted from a cell, such as a mammalian mammaryepithelial cell. In some embodiments a 3′-sequence, which can be derivedfrom a naturally secreted milk protein, can be added to improvestability of mRNA.

In some embodiments, to produce primary cell lines containing aconstruct (e.g., encoding AAT) for use in producing transgenic goats bynuclear transfer, the constructs can be transfected into primary goatskin epithelial cells, which are expanded and fully characterized toassess transgene copy number, transgene structural integrity andchromosomal integration site. As used herein, “nuclear transfer” refersto a method of cloning wherein the nucleus from a donor cell istransplanted into an enucleated oocyte.

Cloning will result in a multiplicity of transgenic animals—each capableof producing an AAT or other gene construct of interest. The productionmethods include the use of the cloned animals and the offspring of thoseanimals. Cloning also encompasses the nuclear transfer of fetuses,nuclear transfer, tissue and organ transplantation and the creation ofchimeric offspring. One step of the cloning process comprisestransferring the genome of a cell, e.g., a primary cell that containsthe transgene of interest into an enucleated oocyte. As used herein,“transgene” refers to any piece of a nucleic acid molecule that isinserted by artifice into a cell, or an ancestor thereof, and becomespart of the genome of an animal which develops from that cell. Such atransgene may include a gene which is partly or entirely exogenous(i.e., foreign) to the transgenic animal, or may represent a gene havingidentity to an endogenous gene of the animal. Suitable mammalian sourcesfor oocytes include goats, sheep, cows, pigs, rabbits, guinea pigs,mice, hamsters, rats, non-human primates, etc. Preferably, oocytes areobtained from ungulates, and most preferably goats or cattle. Methodsfor isolation of oocytes are well known in the art. Essentially, theprocess comprises isolating oocytes from the ovaries or reproductivetract of a mammal, e.g., a goat. A readily available source of ungulateoocytes is from hormonally-induced female animals. For the successfuluse of techniques such as genetic engineering, nuclear transfer andcloning, oocytes may preferably be matured in vivo before these cellsmay be used as recipient cells for nuclear transfer, and before theywere fertilized by the sperm cell to develop into an embryo. MetaphaseII stage oocytes, which have been matured in vivo, have beensuccessfully used in nuclear transfer techniques. Essentially, maturemetaphase II oocytes are collected surgically from either non-superovulated or super ovulated animals several hours past the onset ofestrus or past the injection of human chorionic gonadotropin (hCG) orsimilar hormone.

Thus, in one aspect the disclosure provides mammary gland epithelialcells that produce the AAT disclosed herein. In some embodiments, themammary epithelial cells above are in a transgenic non-human mammal. Insome embodiments, the transgenic non-human mammal is a goat.

Transgenic Animals

In one aspect, the present disclosure also provides a method ofgenerating a genetically engineered or transgenic mammal, by which adesired gene is inserted in the pronucleus of a pre-implantation enbryo.The genetic material integrates into the genome and the resulting animalcarries the genetic material in its genome. In this case the transgeneprovides the genetic information for expression of the recombinant AATinto the milk of the lactating female.

In one aspect, the present disclosure also provides a method of cloninga genetically engineered or transgenic mammal, by which a desired geneis inserted, removed or modified in the differentiated mammalian cell orcell nucleus prior to insertion of the differentiated mammalian cell orcell nucleus into the enucleated oocyte.

In one aspect, the present disclosure also provides mammals obtainedaccording to the methods provided herein, and the offspring of thosemammals. In some embodiments, the present disclosure is used forgenerating caprines or bovines, but the methods can be used with anynon-human mammalian species. The present disclosure further provides forthe use of nuclear transfer fetuses and nuclear transfer and chimericoffspring in the area of cell, tissue and organ transplantation.

Suitable mammalian sources for embryos and oocytes include goats, sheep,cows, pigs, rabbits, guinea pigs, mice, hamsters, rats, primates, etc.,Preferably, in some embodiments, the oocytes are obtained fromungulates, and most preferably, in some embodiments, goats or cattle.Methods for isolation of oocytes are well known in the art. Essentially,oocytes are isolated from the ovaries or reproductive tract of a mammal,e.g., goat. A readily available source of ungulate oocytes is fromhormonally induced female animals.

For the successful use of techniques such as genetic engineering,nuclear transfer and cloning, oocytes may preferably be matured in vivobefore these cells may be used as recipient cells for nuclear transfer,and before they are fertilized by the sperm cell to develop into anembryo. Metaphase II stage oocytes, which have been matured in vivo,have been successfully used in nuclear transfer techniques. Essentially,mature metaphase II oocytes are collected surgically from eithernon-super ovulated or super ovulated animals several hours past theonset of estrus or past the injection of human chorionic gonadotropin(hCG) or similar hormone.

Moreover, it should be noted that the ability to modify animal genomesthrough transgenic technology offers new alternatives for themanufacture of recombinant proteins optimized for use as a therapeuticin humans in terms of their glycan profile. The production of humanrecombinant pharmaceuticals in the milk of transgenic farm animalssolves many of the problems associated with microbial bioreactors (e.g.,lack of post-translational modifications, improper protein folding, highpurification costs) or animal cell bioreactors (e.g., high capitalcosts, expensive culture media, low yields). The current inventionenables the use of transgenic production of biopharmaceuticals,transgenic proteins, plasma proteins, and other molecules of interest inthe milk or other bodily fluid (e.g., urine or blood) of transgenicanimals transgenic for a desired gene that then optimizes theglycosylation profile of those molecules.

A DNA sequence which is suitable for directing production to the milk oftransgenic animals carries a 5′-promoter region derived from anaturally-derived milk protein and is consequently under the control ofhormonal and tissue-specific factors. Such a promoter should thereforebe most active in lactating mammary tissue. According to the currentinvention the promoter so utilized are followed by a DNA sequencedirecting the production of a protein leader sequence which would directthe secretion of the transgenic protein across the mammary epitheliuminto the milk. At the other end of the transgenic protein construct asuitable 3′-sequence, preferably also derived from a naturally secretedmilk protein, may be added to improve stability of mRNA. Examples ofsuitable control sequences for the production of proteins in the milk oftransgenic animals are those from the caprine beta casein promoter.

The production of transgenic animals can now be performed using avariety including micro-injection and nuclear transfer techniques.

Methods of Production of AAT

In one aspect, the disclosure provides methods for production of AAT. Inone aspect, the disclosure provides a method for producing AATcomprising expressing the AAT in mammary gland epithelial cells of anon-human mammal. In some embodiments, the mammary gland epithelialcells are in culture and are transfected with a nucleic acid thatcomprises a sequence that encodes the AAT. In some embodiments, themammary gland epithelial cells are in a non-human mammal engineered toexpress a nucleic acid that comprises a sequence that encodes AAT in itsmammary gland. In some embodiments, the mammary gland epithelial cellsare goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat,mouse or llama mammary gland epithelial cells. In some embodiments, themammary gland epithelial cells are goat mammary gland epithelial cells.

In one aspect the disclosure provides mammary gland epithelial cellsthat express AAT as disclosed herein.

In one aspect the disclosure provides a transgenic non-human mammalcomprising mammary gland epithelial cells that express AAT as disclosedherein.

In another aspect the disclosure provides a method for the production ofa transgenic AAT the process comprising expressing in the milk of atransgenic non-human mammal AAT encoded by a nucleic acid construct. Insome embodiments, the method for producing AAT comprises:

(a) transfecting non-human mammalian cells with a transgene DNAconstruct encoding AAT;

(b) selecting cells in which said AAT transgene DNA construct has beeninserted into the genome of the cells; and

(c) performing a first nuclear transfer procedure to generate anon-human transgenic mammal heterozygous for AAT and that can express itin its milk.

In another aspect, the disclosure provides a method of:

(a) providing a non-human transgenic mammal engineered to express AAT;

(b) expressing AAT in the milk of the non-human transgenic mammal; and

(c) isolating AAT in the milk.

One of the tools used to predict the quantity and quality of therecombinant protein expressed in the mammary gland is through theinduction of lactation (Ebert KM, 1994). Induced lactation allows forthe expression and analysis of protein from the early stage oftransgenic production rather than from the first natural lactationresulting from pregnancy, which is at least a year later. Induction oflactation can be done either hormonally or manually.

In some embodiments, the compositions of AAT provided herein furthercomprise milk. In some embodiments, the methods provided herein includea step of isolating AAT from the milk of a transgenic animal. Methodsfor isolating proteins from the milk of transgenic mammals are known inthe art and are described for instance in Pollock et al., Journal ofImmunological Methods, Volume 231, Issues 1-2, 10 Dec. 1999, Pages147-157. In some embodiments, the methods provided herein include a stepof purifying the expressed AAT.

In one aspect the disclosure provides a method for the production of AATcomprising expressing in the milk of a transgenic non-human mammal AATby a nucleic acid construct. In one embodiment the mammalian mammaryepithelial cells are of a non-human mammal engineered to express the AATin its milk. In some embodiments, the mammalian mammary epithelial cellsare mammalian mammary epithelial cells in culture.

In another embodiment the method comprises:

(a) providing a non-human transgenic mammal engineered to express AAT,

(b) expressing the AAT in the milk of the non-human transgenic mammal;

(c) isolating the AAT expressed in the milk.

In yet another embodiment the method comprises: producing AAT in mammarygland epithelial cells such that the AAT has a high level ofdeoxyhexose. In some embodiments, this method is performed in vitro. Inother embodiments, this method is performed in vivo, e.g., in themammary gland of a transgenic goat.

In some embodiments the methods above further comprise steps forinducing lactation. In some embodiments the methods further compriseadditional isolation and/or purification steps. In some embodiments themethods further comprise steps for comparing the glycosylation patternof recombinantly produced AAT with plasma-derived AAT. In furtherembodiments, the methods further comprise steps for comparing theglycosylation pattern of recombinantly produced AAT to plasma-derivedAAT.

In some embodiments, the methods further include a step of sialylatingthe glycopeptides of AAT.

In some embodiments, the method further comprises comparing thepercentage of deoxyhexose glycosylation present in a population ofrecombinantly produced AAT to the percentage of deoxyhexoseglycosylation in a population of plasma-derived AAT. Experimentaltechniques for assessing the glycosylation pattern of AAT can be any ofthose known to those of ordinary skill in the art or as provided herein,such as below in the Examples. Such methods include, e.g., liquidchromatography mass spectrometry, tandem mass spectrometry, and Westernblot analysis.

Recombinantly produced AAT can be obtained, in some embodiments, bycollecting the AAT from the milk of a transgenic animal produced asprovided herein or from an offspring of said transgenic animal. In someembodiments the AAT produced by the transgenic mammal is produced at alevel of at least 1 gram per liter of milk produced. In someembodiments, the goats expressing rhAAt are produced usingmicroinjection methods.

Methods of Treatment, Pharmaceutical Compositions, Dosage, andAdministration

In one aspect the disclosure provides method of administering acomposition of AAT to a subject in need thereof. In some embodiments theAAT is recombinantly produced. In some embodiments, the AAT is producedin non-human mammary epithelial cells. In some embodiments, the AAT hasa high level of deoxyhexose glycosylation. In some embodiments, the AAThas a high level of sialylation on the AAT-glyco-motifs. In someembodiments, the AAT has a high level of deoxyhexose glycosylation and ahigh level of sialylation on the ATT-glyco-motifs.

In one aspect the disclosure provides methods of administering acomposition of AAT to a subject in need thereof. In some embodiment, thesubject has alpha-1-antitrypsin deficiency. In some embodiments, thesubject has an inflammatory disorder or autoimmune disorder. In someembodiment, the inflammatory disorder is emphysema. In some embodiment,the inflammatory disorder or immune disorders include but are notlimited, to adult respiratory distress syndrome, arteriosclerosis,asthma, atherosclerosis, cholecystitis, cirrhosis, Crohn's disease,diabetes mellitus, emphysema, hypereosinophilia, inflammation, irritablebowel syndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,rheumatoid arthritis, scleroderma, colitis, systemic lupuserythematosus, lupus nephritis, diabetes mellitus, inflammatory boweldisease, celiac disease, an autoimmune thyroid disease, Addison'sdisease, Sjogren's syndrome, Sydenham's chorea, Takayasu's arteritis,Wegener's granulomatosis, autoimmune gastritis, autoimmune hepatitis,cutaneous autoimmune diseases, autoimmune dilated cardiomyopathy,multiple sclerosis, myocarditis, myasthenia gravis, pernicious anemia,polymyalgia, psoriasis, rapidly progressive glomerulonephritis,rheumatoid arthritis, ulcerative colitis, vasculitis, autoimmunediseases of the muscle, autoimmune diseases of the testis, autoimmunediseases of the ovary and autoimmune diseases of the eye, acne vulgari,asthma, autoimmune diseases, celiac disease, chronic prostatitis,glomerulonephritis, hypersensitivities, inflammatory bowel diseases,pelvic inflammatory disease, peperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis, and interstitialcystitis.

In one aspect, the disclosure provides methods of reducing elastaseactivity in the lung, comprising administering a composition of AAT to asubject in an amount sufficient to reduce elastase activity in the lung.

In one aspect, the disclosure provides pharmaceutical compositions whichcomprise AAT and a pharmaceutically acceptable vehicle, diluent orcarrier. In some embodiments, the compositions provided herein comprisemilk.

In one aspect, the disclosure provides a method of treating a subject,comprising administering to a subject a composition provided in anamount effective to treat a disease the subject has or is at risk ofhaving. In one embodiment the subject is a human. In another embodimentthe subject is a non-human animal, e.g., a dog, cat, horse, cow, pig,sheep, goat or primate.

According to embodiments that involve administering to a subject in needof treatment a therapeutically effective amount of AAT as providedherein, “therapeutically effective” or “an amount effective to treat”denotes the amount of AAT or of a composition needed to inhibit orreverse a disease condition alleviate or prevent symptom thereof (e.g.,to treat the inflammation). Determining a therapeutically effectiveamount specifically depends on such factors as toxicity and efficacy ofthe medicament. These factors will differ depending on other factorssuch as potency, relative bioavailability, patient body weight, severityof adverse side-effects and preferred mode of administration. Toxicitymay be determined using methods well known in the art. Efficacy may bedetermined utilizing the same guidance. Efficacy, for example, can bemeasured by a decrease in inflammation or symptom thereof. Apharmaceutically effective amount, therefore, is an amount that isdeemed by the clinician to be toxicologically tolerable, yetefficacious.

Dosage may be adjusted appropriately to achieve desired drug (e.g., AAT)levels, local or systemic, depending upon the mode of administration. Inthe event that the response in a subject is insufficient at such doses,even higher doses (or effective higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. Multiple doses per day are contemplated to achieveappropriate systemic levels of AAT. Appropriate systemic levels can bedetermined by, for example, measurement of the patient's peak orsustained plasma level of the drug. “Dose” and “dosage” are usedinterchangeably herein.

In some embodiments, the amount of AAT or pharmaceutical compositionadministered to a subject is 50 to 500 mg/kg, 100 to 400 mg/kg, or 200to 300 mg/kg per week. In one embodiment the amount of AAT orpharmaceutical composition administered to a subject is 250 mg/kg perweek. In some embodiments, an initial dose of 400 mg/kg is administereda subject the first week, followed by administration of 250 mg/kg to thesubject in subsequent weeks. In some embodiments the administration rateis less than 10 mg/min. In some embodiments, administration of the AATor pharmaceutical composition to a subject occurs at least one hourprior to treatment with another therapeutic agent. In some embodiments,a pre-treatment is administered prior to administration of AAT.

In some embodiments, the AAT or composition thereof is administered at adose of 30 mg/kg to about 60 mg/kg.

In some embodiments the compositions provided are employed for in vivoapplications. Depending on the intended mode of administration in vivothe compositions used may be in the dosage forms of solid, semi-solid orliquid such as, e.g., tablets, pills, powders, capsules, gels,ointments, liquids, suspensions, or the like. Preferably, thecompositions are administered in unit dosage forms suitable for singleadministration of precise dosage amounts. The compositions may alsoinclude, depending on the formulation desired, pharmaceuticallyacceptable carriers or diluents, which are defined as aqueous-basedvehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the human recombinant protein ofinterest. Examples of such diluents are distilled water, physiologicalsaline, Ringer's solution, dextrose solution, and Hank's solution. Thesame diluents may be used to reconstitute a lyophilized recombinantprotein of interest. In addition, the pharmaceutical composition mayalso include other medicinal agents, pharmaceutical agents, carriers,adjuvants, nontoxic, non-therapeutic, non-immunogenic stabilizers, etc.Effective amounts of such diluents or carriers are amounts which areeffective to obtain a pharmaceutically acceptable formulation in termsof solubility of components, biological activity, etc. In someembodiments the compositions provided herein are sterile.

Administration during in vivo treatment may be by any number of routes,including oral, parenteral, intramuscular, intranasal, sublingual,intratracheal, inhalation, ocular, vaginal, and rectal. Intracapsular,intravenous, and intraperitoneal routes of administration may also beemployed. The skilled artisan recognizes that the route ofadministration varies depending on the disorder to be treated. Forexample, the compositions or AAT herein may be administered to a subjectvia oral, parenteral or topical administration. In one embodiment, thecompositions or AAT herein are administered by intravenous infusion.

The compositions, when it is desirable to deliver them systemically, maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compositions in water soluble form.Additionally, suspensions of the active compositions may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompositions to allow for the preparation of highly concentratedsolutions. Alternatively, the active compositions may be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. The component orcomponents may be chemically modified so that oral delivery of the AATis efficacious. Generally, the chemical modification contemplated is theattachment of at least one molecule to the AAT, where said moleculepermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the AAT and increase in circulation time in thebody. Examples of such molecules include: polyethylene glycol,copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone andpolyproline. Abuchowski and Davis, 1981, “Soluble Polymer-EnzymeAdducts” In: Enzymes as Drugs, Hocenberg and Roberts, eds.,Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al., 1982,J. Appl. Biochem. 4:185-189. Other polymers that can be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmolecules. For oral compositions, the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the AAT or by release of thebiologically active material beyond the stomach environment, such as inthe intestine.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according tothe present disclosure may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of thecompositions and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery. The compositions can bedelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Contemplated for use inthe practice of this disclosure are a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butnot limited to nebulizers, metered dose inhalers, and powder inhalers,all of which are familiar to those skilled in the art.

Nasal delivery of a pharmaceutical composition disclosed herein is alsocontemplated. Nasal delivery allows the passage of a pharmaceuticalcomposition of the present disclosure to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

The compositions may also be formulated in rectal or vaginalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compositions, in whose preparation excipients and additivesand/or auxiliaries such as disintegrants, binders, coating agents,swelling agents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference. The AAT and optionallyother therapeutics may be administered per se (neat) or in the form of apharmaceutically acceptable salt. When used in medicine the salts shouldbe pharmaceutically acceptable, but non-pharmaceutically acceptablesalts may conveniently be used to prepare pharmaceutically acceptablesalts thereof. Such salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the disclosure contain an effectiveamount of the AAT and, optionally, other therapeutic agents included ina pharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compositions of the presentdisclosure, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The therapeutic agent(s), including specifically but not limited to theAAT may be provided in particles. Particles as used herein include nanoor microparticles (or in some instances larger) which can consist inwhole or in part of the AAT or other therapeutic agents administeredwith the AAT. The particle may include, in addition to the therapeuticagent(s), any of those materials routinely used in the art of pharmacyand medicine, including, but not limited to, erodible, nonerodible,biodegradable, or nonbiodegradable material or combinations thereof. Theparticles may be microcapsules which contain the AAT in a solution or ina semi-solid state. The particles may be of virtually any shape.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. The methods and techniques ofthe present disclosure are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference, in particular for the teaching that isreferenced hereinabove. However, the citation of any reference is notintended to be an admission that the reference is prior art.

EXAMPLES Pharmacokinetic Study of Alpha 1-Antitrypsin (AAT) in Rats

Plasma-derived (pdAAT), recombinantly produced (rhAAT) and sialylatedrecombinantly produced AAT (Neose) (AAT) were labeled with infrared dyeand injected into rats at 3 and 30 mg/kg. Blood concentrations werefollowed by dot-blot and infrared scan analysis of samples taken overtwo hours at which point the animals were sacrificed andbronchial-alveolar lavage (BAL) fluid was collected. BAL samples wererun on SDS-PAGE and the concentration of AAT was quantitated by infraredanalysis and comparison to a standard curve of the starting materialalso run on SDS-PAGE. The data presented herein demonstrate that whilethe level of recombinant AAT was decreased in the blood compared to theplasma derived, the concentrations in the lung were comparable (SeeFIGS. 4, 5 and 7). Also, the sialylation of the recombinant AAT(“neose”) greatly improved the PK profile of rhAAT (See FIG. 6). Thesialylation improves bioavailability but does not seem to interfere withthe ability of the protein to be sequestered by the lung (See e.g., FIG.7). Approximately twice as much sialylated rhAAT was observed in the BALas in the pdAAT treated rats (See FIGS. 4, 5 and 7). Activity of the BALAAT was assessed by the addition of human neutrophil elastase to thesamples and the observation of a shift of the MW of AAT in both thecomplexed (82 kD) and cleaved (47 kD) form on SDS-PAGE (See FIG. 3).

BAL samples were also run in an ELISA for rat GRO/CINC-1, an analog forhuman IL-8, to determine whether there was activation of the immunesystem by the recombinant AAT or sialylated recombinant AAT. Sampleswere diluted 1/10 in dilution buffer and compared to a standard curve. AGRO/CINC-1 assay was used to determine the extent of inflammation in thelungs (See FIG. 2). Low levels of IL-8, and thus low levels ofinflammation, were observed for all samples.

Recombinant AAT and sialylated recombinant AAT are sequestered into thelung. A study was performed at two doses of AAT, 3 and 30 mg/kg and withplasma derived, recombinant and sialylated recombinant AAT. Two ratswere included as mock controls to test for AAT activity in the BAL of anuntreated animal. Each group included two rats. Injection was iv tailvein and blood samples were taken at 0, 5 30, 60 and 120 minutes whenthe rats were sacrificed and bronchial alveolar lavage fluid wascollected by washing the lungs with 5 ml of PBS (See FIGS. 6 and 7).

Prior to the study, sialylated (Neose) recombinant AAT was generated bydialyzing recombinant AAT into HBS and treating for one hour with 50 mUof sialyltransferase 3 (ST3gal3) in 5 mM CMP-Nan. Sialylation of theterminal galactose was evaluated by an acidic shift on an IEF gel to aposition very close to plasma derived AAT. All samples were labeled withIR800Dye CW, a NHS derivative of the infrared dye with absorption at 800nm. Products were evaluated on SDS-PAGE and by anti-elastase activityassay (See FIG. 3). To determine whether the AAT in the BAL fluid wasactive, samples were mixed with 1 microgram of human neutrophilelastase, or AAT activity buffer, and run on SDS-PAGE. Lanes 1-5 showsthe ability of rhAAT to bind elastase in vitro while lanes 7-10 show theability to bind elastase after harvest from BAL.

Rat samples were assayed by diluting two microliters of serum into 200microl of PBS and loading the samples on a piece of Protran 83nitrocelullose with a 96 well vacuum manifold.

The filter was then scanned on an Odyssey infrared scanner at 800 nm. Agrid was applied to the scan and integrated. BAL samples were alsoevaluated by SDS-PAGE. The presence of AAT in the lung was quantitatedby integration of the bands at about the size of the monomer and above.The larger bands are different forms of labeled AAT includingcomplexation with enzymes (See FIGS. 4 and 5).

Results

Pharmacokinetic profiles showed that pdAAT has the slowest clearance andrecombinant AAT the fastest clearance while sialylation (Neose) greatlyreduced the clearance rate of rhAAT (See FIG. 6).

At 3 mg/kg the rats had detectable quantities of AAT in their BAL fluidsamples. SDS-PAGE analysis of the samples demonstrated all forms couldget into the lungs with the Neose treated AAT rat samples had more AATin the lung than the plasma derived. rhAAT was detectable in the lungeven with low levels in the blood (See FIGS. 6 and 7).

At 30 mg/kg, the level of recombinant AAT in BAL was actually threetimes greater than the plasma derived and sialylated recombinant AAT wasmore than 10 times the concentration of pdAAT.

In order to determine if the AAT observed in the lung samples (i.e.,BAL) was active, one microgram of human neutrophil elastase was mixedwith a rat sample and run on SDS-PAGE. All monomer disappeared and movedinto one of three bands, slightly smaller, slightly larger and atapproximately 80 kD, the expected size of an AAT:elastase complex. Thiswas also observed when the starting material was mixed with elastase. Atime course of this experiment demonstrated that the reaction wascomplete by one minute and the amount of each of the three bands did notchange over 36 minutes.

The immunological state of the rat lung samples was examined by assayingfor GRO/CINC-1, the rat analog of IL-8. Again, there was about 2-foldvariation but levels were low in the range of 75 to 160 pg/ml.

The glycosylation pattern of recombinant AAT and plasma AAT was alsoevaluated. The main difference is the lower level of deoxyhexose in theplasma AAT (The results are shown in FIGS. 8 and 9).

The transgenic animals that express rhAAT as described herein wereprepared according to the methods described in U.S. Pat. No. 7,045,676,such methods are incorporated herein by reference.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as an illustration of certain aspects andembodiments of the invention. Other functionally equivalent embodimentsare within the scope of the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. The advantages and objectsof the invention are not necessarily encompassed by each embodiment ofthe invention.

What is claimed is:
 1. A composition comprising alpha-1-antitrypsin(AAT), wherein the AAT is recombinantly produced.
 2. The composition ofclaim 1, wherein the AAT is produced in mammary epithelial cells of anon-human mammal.
 3. The composition of claim 1, wherein the AAT isproduced in a transgenic non-human mammal.
 4. The composition of claim 2or claim 3, wherein the non-human mammal is a goat, sheep, bison, camel,cow, pig, rabbit, buffalo, horse, rat, mouse or llama.
 5. Thecomposition of claim 4, wherein the non-human mammal is a goat.
 6. Thecomposition of any one of claims 1-5, wherein the recombinantly producedAAT has enhanced deoxyhexose glycosylation compared to plasma-derivedAAT.
 7. The composition of any one of claims 1-6, wherein therecombinantly produced AAT has been modified to increase the sialylationon the AAT-glyco-motifs.
 8. A composition comprising AAT wherein the AAThas a high level of deoxyhexose glycosylation.
 9. A compositioncomprising AAT wherein the AAT has a high level of sialylation on theAAT-glyco-motifs.
 10. A composition comprising AAT wherein the AAT has ahigh level of deoxyhexose glycosylation and a high level of sialylationon the AAT-glyco-motifs.
 11. A composition comprising the AAT of any oneof claims 1-10, further comprising milk.
 12. A composition comprisingthe AAT of any one of claims 1-11, further comprising a pharmaceuticallyacceptable carrier.
 13. Mammary gland epithelial cells that produce theAAT of the compositions of any one of claims 1-12.
 14. A transgenicnon-human mammal comprising the mammary gland epithelial cells of claim13.
 15. A method comprising administering the composition of any one ofclaims 1-12 to a subject in need thereof.
 16. The method of claim 15,wherein the subject has alpha-1-antitrypsin deficiency.
 17. The methodof claim 15, wherein the subject has an inflammatory disorder.
 18. Themethod of claim 17, wherein the inflammatory disorder is emphysema. 19.The method of any one of claims 15-18, wherein the composition isadministered at a dose of from 30 mg/kg to about 60 mg/kg AAT.
 20. Themethod of any one of claims 15-19, wherein the composition isadministered intravenously.
 21. The method of any one of claims 15-19,wherein the composition is administered by inhalation.
 22. A method ofreducing elastase activity in the lung, the method comprisingadministering the composition of any one of claims 1-12 to a subject inan amount sufficient to reduce elastase activity in the lung.